Topic 5 Mobile Genetic Elements v2 PDF

Foundations in Pharmacology PHRM2005 Dr. Ricky R Lareu Mobile Genetic Elements Key Concepts Mobile genetic elements Horizontal gene transfer Mutations Learning Outcomes Be able to describe the 3 types of mobile genetic elements: plasmids, bacteriophage and transposable elements Describe the two majo...

Foundations in Pharmacology PHRM2005 Dr. Ricky R Lareu Mobile Genetic Elements Key Concepts Mobile genetic elements Horizontal gene transfer Mutations Learning Outcomes Be able to describe the 3 types of mobile genetic elements: plasmids, bacteriophage and transposable elements Describe the two major sources of mutation in bacteria and the molecular basis of spontaneous mutations Understand the medical importance of bacterial mutation Be able to describe the three types of horizontal gene transfer mechanisms and how they contribute to changes in bacterial genome (mutation): transformation, transduction and conjugation 2 Reading Material Mims’ Medical Microbiology, Chapter 2, pages 11-23 There are very good animations with quizzes at the end 3 Mobile Genetic Elements In addition to a chromosome, bacteria have additional genetic elements which are mobile – move within or between cells 1) Plasmids 2) Bacteriophage 3) Transposons Bacteria have mechanisms for transferring genes between cells - Horizontal gene transfer 1) Transformation, 2) Transduction and 3) Conjugation These genetic elements carry genes that can confer environmental adaptation-survival advantages e.g. metabolise new carbon source, deactivate antimicrobial substances, produce toxins 4 1) Plasmids Plasmids are small circular DNA molecules found in the cytoplasm of many bacteria  not part of the chromosome  extrachromosomal Plasmids are smaller than the chromosome  Small [1.1-15 kb] and large [60-120 kb] vs chromosome [4,000 kb] The replicate independently of the chromosome but use the same enzymes - Autonomous replication Some plasmids can integrate into the chromosome: episome Bacteria can carry from 1 to 1000 copies, depending on the type and size of plasmids Plasmids are a mobile pool of extra genes, up to 100 extra genes 5 Plasmids Carry Genes Genes for plasmid transfer  Conjugation genes (see later slides) Conjugation Antibiotic resistance genes genes  R plasmids e.g. R1 can confer resistance for ampicillin, chloramphenicol, kanamycin, streptomycin and sulphonamides Virulence genes – examples Other Resistance  E. coli enterotoxin gene, causes genes genes diarrhoea  S. aureus enterotoxin and enzymes involved in virulence e.g. haemolysin, fibrolysin Other properties: heavy metal resistance, resistance to bacteriocins, metabolise additional carbon sources 6 2) Bacteriophage Bacteriophages (phage) are viruses that infect bacterial Their replication invariably leads to death of infected bacteria Bacteriophage can have complex structures Two categories: Virulent (lytic) bacteriophages – reproduce and kill bacteria  Virulent or lytic life cycle Temperate bacteriophages – can integrate into chromosome Bacteriophage T4 and delay death  lysogenic life cycle  lysogenic phage DNA incorporates into the bacterium's DNA and become a non-infectious prophage – temporary not lethal to host  May eventually undergo a lytic cycle o To reproduce See next slide 7 Bacteriophage Life Cycles 8 3) Transposable Elements Transposable elements, Transposons, can jump (“transpose”) from one DNA site to another Properties:  Unable to replicate  Mobility mediated by site-specific recombination: enzymes  Transposition can result in duplication or removal  Source of genetic variation and mutation Can carry additional genes  antibiotic resistance genes  disease causing genes for toxins Major factor in antibiotic resistance Can jump into (and out of) plasmids and chromosome, and within a bacteria’s chromosome 9 Insertion Sequences Elements (IS) The simple form of a transposon Structure: only carry genes for transposition (e.g. transposase) and flanked by inverted terminal repeats Significance: can cause mutation if insert into a gene, can insert into plasmid Examples of ISs: E.coli carries 6-10 copies of IS1 and five copies of IS2 and IS3 Transposase Gene Inverted terminal repeats 10 Transposons – Carry Other Genes Transposons ‘proper’ carry extra genes in addition to those needed for transposition The reason they are medically significant is that they can carry antibiotic resistance genes and virulence genes These genes are flanked by IS elements which have the capacity to translocated the transposon e.g. from the chromosome to a plasmid The plasmid can then transfer these genes to another bacteria 11 Mutations in Bacteria Changes in the genome may occur by two processes:  Mutation  Recombination Mutations frequently arise in bacterial populations Most are deleterious leading to bacterial death but some may confer a survival advantage, although rare Mutations  Spontaneous – error in DNA replication process  Chemical mutagens – induced e.g. radiation, chemicals Mutations are expressed in bacteria due to:  Bacteria being haploid i.e. only one copy of a gene  Rapid growth rate Progeny has changed phenotypic characteristic giving advantage e.g. growth advantage, resistance to an antimicrobial substance Medical importance 12 Molecular Basis of Mutations Types of mutations and their consequences  Point mutation – single change in DNA coding sequence  Deletion – can be large, even deletions of whole genes  Insertions or duplications – can disrupt genes Effects vary from none, reduced survival to death; rarely advantageous Animation: Frame-shift mutation – can terminate gene  protein https://www.genome.gov/genetics-glossary/FrameshiftMutation#:~:text=%E2%80%8BFrameshift%20Mutation&text=A%20frames hift%20mutation%20is%20a,in%20groups%20of%20three%20bases Frequency of mutations  Depends upon environmental conditions and the efficiency of the organisms DNA repair mechanisms  Can vary widely (e.g. mutator strains of bacteria) Image copyright © motifolio.com 13 episome Vertical gene transfer Generation 1 Binary fission Generation 2 Binary fission Generation 3 Through normal proliferation Inheritance Bacterial Gene Transfer Horizontal gene transfer Donor cell Generation 1 Recipient cell Recipient cell 14 Image copyright © motifolio.com Horizontal Gene Transfer Bacteria have developed ways of transferring and recombining genetic information between cells  Unidirectional: donor to recipient Gene transfer can also occur between species Allows bacteria to acquire new genes – potential for selective advantage  Increases variation in population (like principle of sexual reproduction) Three mechanisms 1) Transformation – direct uptake of DNA from environment 2) Transduction – DNA transfer by bacteriophage (virus) 3) Conjugation – DNA transfer by bacteria-to-bacteria contact 15 1) Transformation Some bacterial species are able to take up naked DNA from the environment e.g. from other damaged bacteria This ability is called competence and is carried out by specific proteins called competence factors  Must be close homology between DNA fragment and DNA of recipient for homologous recombination process to work (enzyme-driven specific process)  Otherwise foreign DNA degraded  Plasmids, or parts of, may also be taken up through transformation 16 Transformation cont. Natural competence is found in some species e.g. Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhoeae Chemical treatment can also be used to induce competence  Common procedure in lab to alter bacterial genetics Least medically important of the three processes  because DNA vulnerable when naked outside of cell  may be a factor within an individual’s infection Bacterial transformation animations:  Natural: https://www.youtube.com/watch?v=MRBdbKFisgI  Artificially: https://www.youtube.com/watch?v=9Wnd7PchbCw 17 Naked DNA from lysed cell Competent bacterium with receptor for DNA Receptor/DNA DNA binding Transformed Cell Transformation DNA Uptake Cell Division Homologous Recombination Single-strand 18 2) Transduction Transduction is gene transfer from a donor to a recipient by way of a bacteriophage DNA is resistant to environmental damage (e.g. nucleases) because DNA protected by phage protein coat  more efficient that transformation Not all phages can mediate transduction Transduction is usually restricted to same bacterial species  Phage types usually infect one bacterial species Two types:  Generalised  Specialised 19 Transduction - Generalised Fragments of bacterial chromosome or a plasmid may be packaged erroneously into virion from donor bacteria This DNA can then be delivered into another bacteria (recipient)  If DNA fragment possesses areas of close homology, then recombination with recipient chromosome may occur  Note - Plasmids do not require homologous recombination Can occur with virulent (lytic) phage or temperate phage going through a lytic cycle 20 Animation: https://www.youtube.com/wat ch?v=C44ymgwgA-o Adsorption and Penetration Homologous Recombination Replication Generalised Transduction Adsorption and Penetration Maturation Lysis Bacterial Chromosomal DNA Packaged in Phage 21 Transduction - Specialised Specialised transduction occurs with temperate phages  Prophage integrates in the bacterial chromosome The lysogenic cycle is followed by a lytic cycle, where incorrect excision of prophage may take adjacent segment of bacterial DNA. Integration into recipient bacteria and introduces donor DNA Results in high frequency of DNA transfer i.e. all progeny contain same prophage-host DNA 22 Specialised Transduction Virulent phage Donor cell A B D Chromosome D D D Phage DNA A D B d The viral DNA integrates into chormosome A Recipient cell D D d Animation: https://www.youtube.com/watch?v=ZxbPYekSTLg D Image copyright © motifolio.com Transduced cell 23 Significance of Transduction Phage can alter genetic makeup of bacteria Medically important because they can carry genes for toxins and antibiotic resistance Cholera, botulism and diphtheria toxin genes are all carried by bacteriophage – these virulence genes are what make these bacteria so dangerous Lysogenic conversion allows the bacteria to continue to grow (in number) and cause tissue damage, with ‘new’ genes i.e. the genes are expressed into proteins 24 3) Conjugation Gene transfer by direct physical contact between cells  A form of ‘bacterial mating’ (A) Conjugation usually involves transfer of plasmids Donor has F factor (F+)  Fertility factor  Genes for conjugation  Usually found on a plasmid Recipient lacks F factor (F-) Animation: https://www.youtube.com/watch?v=ycgBX mJw83U 25 Conjugation cont. (B) Occasionally plasmid can be integrated into the chromosome, called an episome Hfr strains: for high-frequency transfer and recombination Episome replication and conjugation may sometimes transfer some chromosomal DNA from F+ donor to F- recipient Significance of conjugation Cross species conjugation is (A) common Major mechanism for gene transfer Multiple antibiotic resistance plasmids Rapid spread of drug resistance (B) Animation: https://www.youtube.com/watch?v=bCnv_ssN984 26 Summary of Mobile Genetic Elements Mobile genetic elements are sources of genetic variation for bacteria in addition to mutations – medically significant because bacteria can acquire new virulence and resistance genes These include plasmids, which are independent replication units, bacteriophage, which infect bacteria and transposable elements that can ‘jump’ between bacterial chromosome and plasmids They are transferred in one direction through horizontal gene transfer mechanisms:  Transformation – take up of DNA fragments from environment, need to be similar to integrate (homologous recombination)  Transduction – through bacteriophage infection; incorrect packaging of host DNA can transfer new DNA to recipient bacteria (generalised, specialised)  Conjugation – the direct transfer of plasmids between bacteria, highly efficient and a major mechanism of spread of bacterial virulence and resistance 27 Summary of Gene Expression Regulation Gene Expression is Regulated at Different Levels in Bacteria Transcriptional Control Initiation of Transcription Translational Control Lifespan of mRNA Translation Rate Post-Translational Control Protein activation Feedback Inhibition 28